The present invention relates to identifying interacting regions of transcription factors, and methods for identifying agents which modulate the interactions, useful for affecting gene regulation, for example, in cellular transformation.
Clustered specific DNA binding sites for an array of activating transcription factors, plus proteins that bend DNA to facilitate contact between bound proteins, have been documented for a number of vertebrate genes (15, 21, 25, 37). These composite structures have been called enhanceosomes (8). The TCR-xcex1 (15) and the IFN-xcex2 (25) enhanceosomes, which are assembled in response to dimerization of the T cell receptor or double-stranded RNA, have been most thoroughly explored. Two classes of genes that are very likely dependent upon enhanceosome assembly have received great attention: genes expressed in a tissue-specific manner that acquire multiple binding proteins during development, and genes that are acutely activated by an external stimulus. These latter structures hold appeal for study because they can be examined in cultured cells where induced synchronous changes occur in all the cells under observation, allowing the acute assembly and disassembly of proteins in an enhanceosome to be potentially revealed.
The Stat family of transcription factors (Darnell, 1997; Stark et al., 1998; U.S. application Ser. No. 08/212,185, filed Mar. 11, 1994 and U.S. Pat. No. 5,716,622; all of the foregoing incorporated herein by reference in their entireties) is activated by polypeptide ligands attaching to specific cell surface receptors, and after tyrosine phosphorylation, dimerization and translocation to the nucleus, can participate within minutes in gene activation (11). It seems likely that Stat molecules bind DNA regions where pre-enhanceosome structures exist (26, 27) and that the arrival of activated Stat dimer(s) is key to forming an active enhanceosome (27). Such a possibility is suggested by experiments showing closely spaced binding sites for Stats and other proteins in the response elements for a number of genes (17, 24, 27, 41). Furthermore DNase and permanganate treatment of cell nuclei revealed proteins bound at or near Stat1 sites before polypeptide treatment. This was followed by detection of Stat molecules binding close to the same DNA regions after induction (26).
One intensively studied set of physiologically important genes that are transcriptionally induced in the liver are the xe2x80x9cacute phase response proteinsxe2x80x9d which increase in the wake of bacterial infections and other toxic assaults. IL-6 stimulation of hepatocytes, via the activation of Stat3, is thought to be the main trigger for inducing the acute phase genes (18). One of the best studied enhancers for acute phase response genes is that of the xcex12-macroglobulin enhancer [(20), reviewed in (18)], a DNA fragment 100 bases long with binding sites for both Stat3 (also called GAS site) and for AP-1, which includes members of the Fos, Jun and ATF families of transcription factors. Extracts from liver nuclei of IL-6 treated animals or transformed hepatocytes (hepatoma cells) in culture indicated induced binding to this region. Since Stat3 and c-Jun interacted in yeast 2-hybrid assays and cooperated in maximizing the transcription responses of reporter genes containing the xcx9c100 bp enhancer (30, 31), it seemed likely that this genomic region might form a Stat-dependent enhanceosome.
It is towards identifying particular regions of transcription factor interactions responsible for transcriptional activation, and the use of this information in the design of methods and the subsequent identification of agents capable of modulation the interaction, that the present invention is directed.
In its broadest aspect, the present invention is directed to methods for identifying an agent capable of modulating the interaction between a transcription factor and a Stat protein comprising the steps of
(a) providing said transcription factor or a fragment thereof;
(b) providing a Stat protein fragment comprising a region within from about residue 107 to about residue 377 of the Stat protein;
(c) incubating mixtures of the transcription factor or fragment thereof and the Stat protein fragment with and without said agent;
(d) detecting the extent of interaction between the transcription factor or fragment thereof and the Stat protein fragment in each of the mixtures; and
(e) identifying an agent as capable of modulating said interaction as one which alters the extent of interaction between the transcription factor or fragment thereof and the Stat protein fragment.
The agent may be capable of modulating cellular transformation. The Stat protein fragment of the foregoing method may comprise the coiled-coil domain of the Stat protein and the first three xcex2-strands of the DNA-binding domain of the Stat protein. Non-limiting examples of Stat protein include Stat1, Stat2, Stat3, Stat4, Stat5 or Stat6. For example, for Stat3, fragments may include about residue 107 to about residue 358, about residue 130 to about residue 358, about residue 155 to about residue 377, about residue 193 to about residue 377, about residue 249 to about residue 377, or about residue 282 to about residue 377. Particular suitable fragments include those set forth as SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25. The Stat protein or fragment may be labeled with a detectable label, for example, a GST fusion sequence or an epitope tag.
The transcription factor used in the above-described method may be a member of the JUN, the FOS, or the ATF families of transcription factors. For example, a JUN transcription factor may be c-Jun, JunB and JunD. A FOS transcription factor may be c-Fos, FosB, Fra-1 and Fra-2. An ATF transcription factor may be ATF-1, ATF-2, ATF-3 and ATF-4. These examples are merely illustrative and non-limiting. The transcription factor fragment may include the COOH-terminal region, or the bZIP region.
In one example, the transcription factor is c-Jun. A fragment of c-Jun may include the region of about residue 105 to about residue 334 of c-Jun, or the region of about residue 105 to about residue 263 of c-Jun. The transcription factor or fragment thereof may be labeled with a detectable label, for example, a radiolabel.
The detection of the extent of interaction of the foregoing method may be carried out for example using the techniques of is performed by GST protein association assay, coimmunoprecipitation, eletrophoretic mobility shift assay (EMSA), or the yeast 2-hybrid system.
In one example wherein the Stat protein is Stat3, the agent modulates the interaction between the transcription factor and Stat3 protein at residues of said Stat3 protein such as but not limited to residues 130-154, residues 343-358, and the combination thereof. The agent may be a Stat protein antagonist or agonist. In the example wherein the transcription factor is c-Jun, the modulation of interaction may occur at about residue 105 up to about 334 of c-Jun, about residue 105 up to about 334 of c-Jun, or about residues 105-263 of c-Jun.
In another aspect of the present invention, methods are provided for identifying an agent capable of modulating the transcriptional cooperation between a transcription factor and a Stat protein comprising the steps of:
(a) providing a transiently transfected cell bearing a Stat-inducible reporter gene;
(b) introducing into the cell a transcriptionally cooperative combination of a wild-type Stat protein or mutant thereof, and a wild-type transcription factor or mutant thereof;
(c) inducing the expression of the reporter gene;
(d) determining the extent of expression of the reporter gene in the presence and absence of said agent; and
(e) identifying an agent capable of modulating said interaction as one able to alter the expression of the reporter gene.
The agent is capable of modulating cellular transformation. The Stat protein or mutant thereof comprises the coiled-coil domain of said Stat protein and the first three xcex2-strands of the DNA-binding domain of said Stat protein. Non-limiting examples of Stat proteins suitable for the practice of the foregoing method include Stat1, Stat2, Stat3, Stat4, Stat5 or Stat6.
In the example wherein the Stat protein is Stat3, the agent may modulate the interaction between the transcription factor and said Stat3 protein at residues of the Stat3 protein of residues 130-154, residues 343-358, or the combination. In another example, the Stat3 mutant has at least one mutation in a region of the native Stat3 sequence at positions selected from the group consisting of residues 130-154, residues 343-358, and the combination thereof. Examples of particular mutants include Stat3(L148A) (SEQ ID NO:30), Stat3( V151A) (SEQ ID NO:31), and Stat3(T346A, K348A, R350A) (SEQ ID NO:29).
The Stat protein or mutant thereof is labeled with a detectable label, for example, a GST fusion sequence or an epitope tag.
Transcription factors useful in the above method include but are not limited to members of the JUN, the FOS, and the ATF families of transcription factors. For example, a JUN transcription factor may be c-Jun, JunB and JunD. A FOS transcription factor may be c-Fos, FosB, Fra-1 and Fra-2. An ATF transcription factor may be ATF-1, ATF-2, ATF-3 and ATF-4. The transcription factor or fragment thereof may be labeled with a detectable label, for example, a radiolabel.
In the example wherein the transcription factor is c-Jun, the agent may modulate the transcriptional cooperation between the c-Jun and Stat3 protein at residues of the c-Jun protein at residues 105-334. The c-Jun interaction regions may be within residues about 105 and up to about 334, or residues about 105 to about 263.
In another broad aspect of the present invention, methods are provided for identifying mutants in a transcription factor or Stat molecule, or in both, wherein the mutant is capable of modulating the transcriptional cooperation between the transcription factor and the Stat protein. The method comprises:
(a) providing a transiently transfected cell bearing a Stat-inducible reporter gene;
(b) introducing into the cell a wild-type Stat protein or mutant thereof; and a wild-type transcription factor or mutant thereof, wherein at least one of the introduced Stat protein or transcription factor is mutant;
(c) inducing the expression of said reporter gene;
(e) determining the extent of expression of the reporter gene compared to that extent in a cell having a wild-type form of at least one of the mutant transcription factor or the mutant Stat protein; and
(f) identifying an mutant as one capable of modulating the interaction as one able to alter the expression of the reporter gene.
The Stat protein or mutant thereof may comprise the coiled-coil domain of said Stat protein and the first three xcex2-strands of the DNA-binding domain of said Stat protein. Non-limiting examples of Stat protein include Stat1, Stat2, Stat3, Stat4, Stat5 and Stat6. In the example of Stat3, the mutation may modulate the transcriptional cooperation between the transcription factor and Stat3 at residues of said Stat3 protein such as but not limited to residues 130-154, residues 343-358, and the combination thereof. The Stat3 mutant may have at least one mutation in a region of the native Stat3 sequence at positions within residues 130-154, residues 343-358, or the combination thereof. Particular non-limiting examples include Stat3(L148A) (SEQ ID NO:30), Stat3(V151A) (SEQ ID NO:31), and Stat3(T346A, K348A, R350A) (SEQ ID NO:29).
The Stat protein or mutant thereof may be labeled with a detectable label, such as a GST fusion sequence or an epitope tag.
In the practice of the foregoing method, the transcription factor may be a member of the JUN, the FOS, or the ATF families of transcription factors. For example, a JUN transcription factor may be c-Jun, JunB and JunD. A FOS transcription factor may be c-Fos, FosB, Fra-1 and Fra-2. An ATF transcription factor may be ATF-1, ATF-2, ATF-3 and ATF-4. The transcription factor or fragment thereof may be labeled with a detectable label, for example, a radiolabel.
In the example of c-Jun and a Stat protein, the mutation may modulate the transcriptional cooperation between c-Jun and the protein at residues of said c-Jun at positions about 105 up to about 334, or about 105 to about 263.
The invention is also directed to polynucleotides encoding the various aforementioned Stat3 fragments, and the Stat3 mutants Stat3(L148A) (SEQ ID NO:30), Stat3(V151A) (SEQ ID NO:31), or Stat3(T346A, K348A, R350A) (SEQ ID NO:29). It is also directed to such polynucleotides which include a GST fusion sequence or an epitope tag.
The invention is further directed to cells transiently expressing a mutant Stat3 protein, the mutant Stat3 proteins as described above.
The invention is also directed to fragments of c-Jun 1-104 (SEQ ID NO:26) or 105-334 (SEQ ID NO:27), their polynucleotide sequences, as well as cells transiently expressing a mutant c-Jun fragment as described above.
The invention is also directed to methods for identifying a mutant Stat protein capable of modulating the transcriptional cooperation between a Stat protein and a transcription factor comprising the steps of:
(a) providing a transformed cell line;
(b) transfecting the transformed cell line with a Stat mutant suspected of interfering with the interaction between said Stat and a transcription factor;
(c) examining the transfected cell line for evidence of alteration of transformation in contrast to said cell line transfected with the wild-type Stat; and
(d) identifying a mutant capable of modulating the transcriptional cooperation between a Stat protein and a transcription factor as one which alters the transformation of the cells.
For example, evidence of alteration of transformation may be a change in morphology on soft agar.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.